Nm23 gene has been reported to encode proteins involved in the development and differentiation of a normal tissue, and its expression is decreased in various metastatic cells. Nm23 proteins belong to a tumor metastasis suppressor and generally consist of 150 to 180 amino acids. Nm23 proteins contain a leucine zipper motif and exhibit nucleoside diphosphate kinase (NDPK) activity. Two human Nm23 homologues, Nm23-H1 and Nm23-H2, consist of 152 amino acids having molecular weights of 17,143 and 17,294, respectively. In particular, it has been found that Nm23-H1 plays an important role in tumor metastasis and other various cell mechanisms including cell proliferation, embryogenesis, differentiation and tumorigenesis.
The mechanisms by which Nm23 affects tumor development and metastasis have not yet been clearly investigated. NDPK transfers a phosphoryl group between nucleoside triphosphate and nucleoside diphosphate via a covalent phosphoenzyme intermediate. For such a phosphorylation, histidine 118 of each Nm23-H1 and Nm23-H2 is served as a target site. Apart from the NDPK-mediated histidine phosphorylation, serine autophosphorylation was observed in Nm23 (MacDonald N J et al., J. Biol. Chem. 268: 25780-25789, 1993). When melanoma cells of Nm23 transfected mice were compared with a control cell, there was a direct correlation between the in vivo phosphorylation level of Nm23 serine and the inhibition of tumor metastatic potential. Serine phosphorylation of mouse Nm23 is inhibited by cAMP in vivo, while by forskolin in vitro, which demonstrates that the phosphorylation is controlled by signal transduction pathways.
Initially, Nm23 expression was reported to closely correlate to mouse melanoma cell lines with poor metastatic potential. The relationship between Nm23 reduced expression and tumor metastasis has been regarded as direct evidence to support the fact that Nm23 functions as a tumor metastasis suppressor (Steeg, P. S., Breast Dis. 10: 47-50, 1998). Inducible overexpression of Nm23 exhibited remarkably reduced metastatic potential in a highly metastatic cancer cell line. The Nm23 gene cloned as a putative tumor metastasis suppressor gene exhibits serine/threonine specific phosphotransferase and histidine protein kinase activities, as well as NDPK activity. Further, the expression of Nm23 is reduced as hematopoietic stem cells (HSC) are differentiated, which suggests that Nm23 is an important factor for anti-differentiation in those cells (Gervasi, F. et al., Cell Growth Differ 7: 1689-95, 1996). It has been found that Nm23 exhibits a strong inhibitory effect on tumor metastasis upon temporary transfection in forced inducible gene expression and in vitro metastasis model systems of human tumors (Hirayama, R. et al., J. Natl. Cancer Inst. 83: 1249-50, 1991; Nakayama, T. et al., J. Natl. Cancer Inst. 84: 1349-54, 1992; Leone, A. et al., Oncogene 8: 2325-33, 1993; Leone, A. et al., Oncogene 8: 855-65, 1993). In contrast, Nm23 mutation, leading to the loss of NDPK activity, had no influence on the inhibitory function of Nm23 in breast cancer cells (MacDonald, N. J. et al., J. Biol. Chem. 271:25107-16, 1996).
The most authentic evidence for the fact that Nm23 is a metastasis suppressor is revealed when the Nm23 gene is transfected into tumor cell lines. In metastatically competent cells, the administration of high dose Nm23 showed reduced metastatic activity by 40 to 98% as compared with a control transfectant (Leone, A. et al, Cell 65: 25-35, 1991; Leone, A. et al., Oncogene 8:2325-33, 1993).
Recently, it has been reported that Nm23 interacts with a kinase suppressor of Ras (KSR) discovered in the Drosophilae (Drosophilar melanogaster) and nematode (Caenorhabditis elegans) systems (Morrison, D. K., J. Cell Sci. 114: 1609-12, 2001). KSR is a scaffold protein of a mitogen-activated protein kinase (MAPK) cascade (Burack, W. R. and Shaw, A. S., Curr. Opin. Cell Biol. 12: 211-6, 2000; Pawson, T. and Scott, J. D., Science 278: 2075-80, 1997). Such scaffold protein is necessary to enhance the rate of phosphorylation and contribute to the specificity and stabilization of the phosphorylation pathway. Once the MAPK signal transduction pathway is activated by active Ras, KSR is forcedly dephosphorylated, and then, serve as a scaffold for the activation of MAPK cascade. During this process, Nm23 phosphorylates KSR serine 392, which is a binding site for another associated protein of KSR. If the serine 392 is mutated, Nm23 phosphorylates KSR serine 434. The metastatic inhibitory activity of Nm23 has been clearly demonstrated by the fact that metastatic potential is inhibited in various tumor cells transfected with Nm23 gene (Yoshida, T. et al., J Gastroenterol. 35: 768-74, 2000). In cells activated by the stimulation of the MAPK cascade, the interaction between Nm23 and KSR induces KSR phosphorylation in vitro in a complicated manner via a histidine-dependent pathway (Hartsough, M. T. et al., J. Biol. Chem. 277: 32389-99, 2002). Further, the in vivo association of KSR and Nm23 inhibits the phenotypic effect of active Ras which activates the MAPK cascade.
Accordingly, the administration of high dose Nm23 protein may phosphorylate and inactivate KSR in vivo, leading to the inhibition of Ras-mediated MAPK cascade. The present inventors have therefore believed that the inhibition of MAPK signal transduction pathway mediated by re-phosphorylation of KSR may inhibit cell proliferation, differentiation and migration of cancer cells and exhibit anti-metastatic effect in various human cancers, and endeavored to develop new anti-metastatic agents by using the Nm23 protein.
Meanwhile, small molecules derived from synthetic compounds or natural compounds can be transported into the cells, whereas macromolecules, such as proteins, peptides, and nucleic acids, cannot. It is widely understood that macromolecules larger than 500 kDa are incapable of penetrating the plasma membrane, i.e., the lipid bilayer structure, of live cells. To overcome this problem, a macromolecule intracellular transduction technology (MITT) was developed (Jo et al., Nat. Biotech. 19: 929-33, 2001), which allows the delivery of therapeutically effective macromolecules into cells, making the development of new drugs using peptides, proteins and genetic materials possible. According to this method, if a target macromolecule is fused to a hydrophobic macromolecule transduction domain (MTD) and other cellular delivery regulators, synthesized, expressed, and purified in the form of a recombinant protein, it can penetrate the plasma membrane lipid bilayer of the cells, be accurately delivered to a target site, and then, effectively exhibit its therapeutic effect. Such MTDs facilitate the transport of many impermeable materials which are fused to peptides, proteins, DNA, RNA, synthetic compounds, and the like into the cells.
Accordingly, the inventors of the present invention have developed a method of mediating the transport of a metastasis suppressor Nm23 into the cells, where cell permeable Nm23 recombinant proteins are engineered by fusing a MTD to the metastasis suppressor Nm23. Such cell permeable Nm23 recombinant proteins have been found to efficiently mediate the transport of a metastasis suppressor Nm23 into the cells in vivo as well as in vitro and can be used as anti-metastatic agents for inhibiting metastasis occurring in various human cancers.